12 research outputs found

    Establishment of hICD-expressing stable cells.

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    <p>(A) Transient expression of hTMC construct in HEK293 cells produced, in addition to the expected TMC fragment, a band similar in size to the intracellular domain of FPC. * Ig heavy and light chains. (B) Western blotting showing the levels of hICD in four mIMCD-3 cell lines stably expressing hICD (hICD3, 5, 6, 13), along with four empty vector control cell lines (CTL1, 2, 3, 4). IB, immunoblotting. (C) FPC C-tail was mainly localized in the nuclei of mIMCD-3 cells (hICD3) compared to the control cell line (CTL3). IF, immunofluorescence. Scale bar, 5 µm.</p

    mTOR activation in hICD-expressing cells and ARPKD kidneys.

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    <p>(A) In contrast to that in control cells, pS6K1<sup>T389</sup>, pS6<sup>S235/236</sup>, pS6<sup>S240/244</sup>, and pAkt<sup>S473</sup> were activated and p4E-BP1<sup>T37/46</sup> was suppressed in serum-starved hICD-expressing cells (DMEM/F12/0.5% serum for 16 h). (B) In amino acid-starved hICD-expressing cells, Akt<sup>S473</sup> and S6K1<sup>T389</sup> were hyperphosphorylated compared to those in controls, whereas 4E-BP1<sup>T37/46</sup> was de-phosphorylated. The bar graphs represented the activation of specified proteins expressed as a ratio of the phosphorylation form/total protein (N = 3). (C) In two pairs of FPC knockdown cells (2850S1 and 2141S26), S6K1<sup>T389</sup> was hyperphosphorylated under serum-starved conditions. The numbers on the Y-axis were arbitrary units of the bands intensity ratio. (D) S6K1<sup>T389</sup> was highly phosphorylated while phosphorylation of 4E-BP1<sup>T37/46</sup> was reduced in ARPKD kidneys (AR-1, 2, 3), compared to normal controls (CTL-1, -2, -3). Open bars, control cells; filled bars, hICD-expressing cells. Values were expressed as mean ± SD. * p<0.05, ** p<0.01, *** p<0.001.</p

    Opposite roles of full-length FPC and C-tail on mTOR.

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    <p>(A) Human full-length FPC- (hFPCL)- and control empty vector (P4L)-expressing LLC-PK1 cells were transfected with various amounts of hICD plasmid DNA (0, 1, 3 µg). Twenty-four hours after transfection, cells were serum starved for 24 h before protein extraction. Expression of full-length FPC inhibited the phosphorylation of p70S6K<sup>T389</sup> and pS6<sup>S240/244</sup> in comparison to the controls, while hICD expression antagonized the inhibition of full-length FPC on p70S6K<sup>T389</sup> and pS6<sup>S240/244</sup> in a dose-dependent fashion. One representative experiment was shown.</p

    FPC C-tail expression caused cystogenesis in 3D culture.

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    <p>hICD cells from all four cell lines (3, 5, 6, 13), in parallel with cells from four control lines, were cultured in collagen I gels. All hICD-expressing lines (3, 6, 13, 100%; 5, <100%) formed cyst-like structures in contrast to tubule-like structures formed by control cells. The representative pictures were presented (A, B). (A) Tubulogenesis of both control (CTL1, CTL2) and hICD (hICD3, hICD6) cells at 1, 3 and 7 days after seeding in collagen I gels. At day 3 and 7, in contrast to control cells, which developed tubule-like structures (100%), hICD-expressing cells formed cyst-like structures (100%). (B) Confocal microscopy reveals the presence of a lumen in the tubule-like structure developed from control cells (CTL1, 2) and a central cavity in cyst-like structures in hICD-expressing cells (CTL3, 6) after 8-day culture. A segment of a tubule was shown with a higher magnification. HGF was not used in this experiment. Red, rhodamine phalloidin; green, acetylated α-tubulin; blue, DAPI.</p

    Decreased cell proliferation rate and increased apoptosis in hICD-expressing cells.

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    <p>(A) The number of hICD-expressing cells was significantly lower than that of control cells when cultured in 1% but not in 10% serum-containing medium (N = 3 hICD3, 6, 13). One representative of three experiments was presented. (B) With flow cytometry analysis, more hICD-expressing cells passed the restriction point in G1/0-phase but were arrested in S-phase after serum removal for 48 h. Addition of 10% serum for 24 h completely compromised the effect of hICD expression on the cell cycle profile (N = 3 hICD3, 6, 13). One representative of three experiments was presented. (C) More apoptotic hICD-expressing cells were found in the absence of serum by flow cytometry analysis (N = 3 hICD3, 6, 13). One representative of at least three experiments was presented. (D) Three control and three hICD cell lines were used for this experiment. Pictures from two control and hICD cell lines were presented. TUNEL staining assay confirmed an increase of apoptotic hICD-expressing cells. Bar graph represented statistical significance (N = 200 cells from three controls and hICD cell lines). Open bars, control cells; filled bars, hICD-expressing cells. Values were expressed as mean ± SD. * p<0.05, ** p<0.01, *** p<0.001. Scale bar, 10 µm.</p

    Cystin has a functional nuclear localization signal at the N-terminus.

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    <p>The subcellular localization of cystin and its variants was determined by immunoblotting. The wild-type cystin<sub>wt</sub>, the N-terminal deletion mutant cystin<sub>▵1-20</sub>, and the site-directed mutant cystin<sub>G2A</sub>, each tagged with GFP were stably-transfected in mIMCD-3 cells. (<b>A</b>) The distribution of wild-type cystin<sub>wt</sub>::GFP in the four fractions prepared from mIMCD-3 cells is shown by western blot (above) and using densitometry (below). (<b>B</b>) The distribution for cystin<sub>▵1-20</sub>::GFP is similarly shown by western and densitometry. (<b>C</b>) The distribution of the site-directed mutant cystin<sub>G2A</sub>::GFP is shown in the four fractions of mIMCD-3 cells using western blot analysis and densitometry. (<b>D</b>) To demonstrate the reliability of the fractionation protocol, wild type mIMCD-3 cells were fractionated (n = 5 experiments) and 10 µg of each fraction was analyzed by western blotting with fraction specific marker as indicated. The fractions are cytosol (1), membrane (2), nuclear (3) and cytoskeleton (4).</p

    An interaction between cystin and necdin was identified in a yeast two-hybrid screen.

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    <p>(<b>A</b>) After the initial unbiased screen full-length cystin was cloned into a prey vector, while necdin was cloned into a bait vector. Both were co-transformed and spread onto control SD/-Leu-Trp (data not shown) and selective SD/-Ade-His-Leu-Trp plates. A series of deleted cystin constructs were co-transformed with full-length necdin to isolate the necdin-interacting domain. Y2H positive (pGADT7-LgT+pGBKT7-p53) and negative controls (pGADT7-LgT+pGBKT7-Lam) are also shown. A scheme of the interactions between the versions of cystin and necdin are shown at the lower panel. The C-terminal 25 aa of cystin are required for its interaction with necdin. (<b>B</b>) A series of necdin constructs (prey) were co-transformed along with full-length cystin (bait) to determine the cystin-interacting domain. A scheme of the interaction between the versions of necdin and cystin are shown in the lower panel. Necdins cystin interaction domain lies within aa 71 - 305.</p

    Endogenous cystin and necdin were expressed in mIMCD-3 cells.

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    <p>(<b>A</b>) RT-PCR was performed to detect the expression of cystin and necdin in mIMCD-3 cells. Total RNA was isolated from confluent mIMCD-3 cells and equal amounts of cDNA were used as a template for RT-PCR. Primer pairs for cystin and necdin were designed to amplify 207 bp and 632 bp target bands, respectively. β-actin was used as a positive control; water was used as a negative control. The PCR products were excised from the gel, purified, and verified by sequencing (data not shown). (<b>B</b>) Western blot analysis of endogenous cystin in confluent mIMCD-3 cells. The cells were fractioned to cytoplasmic, membrane, and nuclear fractions. Analysis of control proteins for the different fractions were shown in the lower panel. Endogenous cystin migrates at 25 kDa and accumulated in the membrane fraction. <b>(C)</b> Western blot analysis of endogenous necdin in confluent mIMCD-3 cells. Endogenous necdin was detected in the nuclear fraction and migrated at 40 kD when probed with a necdin N-terminal antibody (N20, arrowhead). Western blot analysis of Myc-necdin in mIMCD-3 cells using a myc antibody also reveals a band at 40 kD (right). The band at ∼30 kDa is an unidentified artifact of the antibody used <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083062#pone.0083062-DeCamilli1" target="_blank">[103]</a>.</p

    The interaction between cystin and necdin was further verified by GST pull-down and co-immunoprecipitation.

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    <p>(<b>A</b>) GST pull-down was used to verify the interaction between cystin and necdin. pCMV-GST-cystin and pCMV-myc-necdin were co-transfected into COS-7 cells. The precipitated samples were run on a PAGE gel and probed with myc (lanes 1-4) and GST antibodies (lanes 5-8). Whole cell lysate was used to assess protein expression (lane 1-2 and 5-6). Compared to the negative control (lane 3), the specific interaction between cystin and necdin is clear (lane 4, arrowhead). (<b>B</b>) Co-IP was used to verify the interaction between cystin and necdin. pCMV-HA-cystin and pCMV-myc-necdin were co-transfected into COS-7 cells. The protein complex of HA-cystin and myc-necdin in the cell lysate was precipitated using the HA tag, and the samples were probed with myc (lanes 1-4) and cystin antibodies (lanes 5-8), respectively. Whole cell lysate was used to control for protein expression (lane 1-2 and 5-6). Compared with the negative control (lane 3), a specific band indicates a physical interaction between cystin and necdin (lane 4, arrowhead).</p

    Vector switch to eliminate false positive clones.

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    <p>+LgT and Lam+LgT were used as positive and negative controls, respectively. Necdin was switched from the prey vector (pGADT7) to the bait vector (pGBKT7). The new constructs were re-tested and their interactions were validated. 3-AT: 3-amino-1,2,4-triazole, a competitive inhibitor of the His3 protein.<sup></sup> p53</p
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